This invention relates to the new amino acid sequence of the malate:quinone oxidoreductase enzyme protein (Mqo) of Enterobacteriaceae and to a process for the fermentative preparation of L-threonine using Enterobacteriaceae in which the mqo gene is enhanced.
L-Threonine is used in animal nutrition, in human medicine and in the pharmaceuticals industry. It is known that L-threonine can be prepared by fermentation of strains of Enterobacteriaceae, in particular Escherichia coli and Serratia marcescens. Because of their great importance, work is constantly being undertaken to improve the preparation processes. Improvements to the process can relate to fermentation measures, such as e.g. stirring and supply of oxygen, or the composition of the nutrient media, such as e.g. the sugar concentration during the fermentation, or the working up to the product form by e.g. ion exchange chromatography, or the intrinsic output properties of the microorganism itself.
Methods of mutagenesis, selection and mutant selection are used to improve the output properties of these microorganisms. Strains which are resistant to antimetabolites, such as e.g. the threonine analogue xcex1-amino-xcex2-hydroxyvaleric acid (AHV), or are auxotrophic for metabolites of regulatory importance and produce L-threonine are obtained in this manner.
Methods of the recombinant DNA technique have also been employed for some years for improving the strain of Enterobacteriaceae strains which produce L-threonine, by amplifying individual threonine biosynthesis genes and investigating the effect on the L-threonine production.
The inventors had the object of providing new measures for improved fermentative preparation of L-threonine.
The invention provides a polypeptide from Enterobacteriaceae with malate:quinone oxidoreductase (Mqo) activity (E.C. 1.1.99.16) chosen from the group consisting of
a) polypeptide with the amino acid sequence shown in SEQ ID NO. 2, or
b) polypeptide which is at least 70%, preferably at least 80%, particularly preferably at least 90 to 95% identical to the amino acid sequence shown in SEQ ID NO. 2, or
c) polypeptide according to SEQ ID NO. 2, including deletion, insertion or exchange of one or more amino acids, or
d) polypeptide according to SEQ ID NO. 2, including N- or C-terminal lengthening by one or more amino acids,
the total length of the polypeptide according to b), c) or d) being at least 514 and at most 544, preferably at least 519 and at most 539, in a preferred form at least 524 and at most 534, particularly preferably at least 527 and at most 531 amino acid radicals.
The invention furthermore provides a polynucleotide from Enterobacteriaceae which codes for a polypeptide with malate:quinone oxidoreductase (Mqo) activity (E.C. 1.1.99.16), chosen from the group consisting of
a) DNA which contains the nucleotide sequence corresponding to nucleobases 7 to 1593 of SEQ ID NO. 1, or
b) DNA according to a) corresponding to the degeneration of the genetic code, or
c) DNA according to a) containing sense mutations of neutral function, or
d) DNA which is at least 70%, preferably at least 80%, particularly preferably at least 90 to 95% identical to that mentioned in a) or b), or
e) polynucleotide which hybridizes with the DNA according to a), b), c) or d).
The invention also provides
a DNA which is capable of replication and codes for the polypeptide shown in SEQ ID NO. 2,
a vector containing the mqo gene corresponding to nucleobases 7 to 1593 of SEQ ID NO. 1, in particular plasmid pMW218mqo shown in FIG. 1.
xe2x80x9cPolynucleotidexe2x80x9d in general relates to polyribonucleotides and polydeoxyribonucleotides, it being possible for these to be non-modified RNA or DNA or modified RNA or DNA.
xe2x80x9cPolypeptidesxe2x80x9d is understood as meaning peptides or proteins which comprise two or more amino acids bonded via peptide bonds.
The polypeptides according to the invention include the polypeptides according to SEQ ID NO. 2, which have malate:quinone oxidoreductase activity, and also those which are at least 70%, preferably at least 80% and in particular at least 90% to 95% identical to the polypeptide according to SEQ ID NO. 2 and have the activity mentioned.
Finally, the invention provides a process for the fermentative preparation of L-threonine using Enterobacteriaceae which in particular already produce L-threonine and in which the nucleotide sequence(s) which code(s) for the mqo gene are enhanced, in particular over-expressed.
In particular, the process is a process for the preparation of L-threonine, which comprises carrying out the following steps:
a) fermentation of microorganisms of the family Enterobacteriaceae in which at least the mqo gene is enhanced (over-expressed), optionally in combination with further genes,
b) concentration of the L-threonine in the medium or in the cells of the microorganisms of the family Enterobacteriaceae, and
c) isolation of the L-threonine.
The term xe2x80x9cenhancementxe2x80x9d in this connection describes the increase in the intracellular activity of one or more enzymes or proteins in a microorganism which are coded by the corresponding DNA, for example by increasing the number of copies of the gene or genes, using a potent promoter or a gene which codes for a corresponding enzyme or protein with a high activity, and optionally combining these measures.
The microorganisms which the present invention provides can prepare L-threonine from glucose, sucrose, lactose, fructose, maltose, molasses, starch, or from glycerol and ethanol. They are representatives of Enterobacteriaceae, in particular of the genera Escherichia and Serratia. Of the genus Escherichia the species E. coli and of the genus Serratia the species Serratia marcescens are to be mentioned in particular.
Suitable L-threonine-producing strains of the genus Escherichia, in particular of the species E. coli, are, for example
Escherichia coli TF427
Escherichia coli H4578
Escherichia coli KY10935
Escherichia coli EL1003
Escherichia coli VNIIgenetika MG-442
Escherichia coli VNIIgenetika VL334/pYN7
Escherichia coli VNIIgenetika M1
Escherichia coli VNIIgenetika 472T23
Escherichia coli VNIIgenetika TDH-6
Escherichia coli BKIIM B-3996
Escherichia coli BKIIM B-5318
Escherichia coli B-3996-C43
Escherichia coli B-3996-C80
Escherichia coli B-3996/pTWV-pps
Escherichia coli B-3996(pMW::THY)
Escherichia coli B-3996/pBP5
Escherichia coli kat 13
Escherichia coli KCCM-10132
Suitable L-threonine-producing strains of the genus Serratia, in particular of the species Serratia marcescens, are, for example
Serratia marcescens HNr21
Serratia marcescens TLr156
Serratia marcescens T2000
The nucleotide sequence of the chromosome of E. coli is known and is available in databanks accessible to the public, such as, for example, the databank of the European Molecular Biology Laboratories (EMBL, Heidelberg, Germany). Examples of such sequences deposited are the entries accessible under number AE000310 or D90850.
In the work on the present invention it was possible to identify the mqo gene, which codes for malate:quinone oxidoreductase, of E. coli (SEQ ID NO. 1) and the amino acid sequence of the Mqo enzyme protein formed (SEQ ID NO. 2).
It has furthermore been possible to isolate two further new malate:quinone oxidreductase [sic] proteins, designated protein B and C, which have the N-terminal amino acid sequence shown in SEQ ID No. 11 and 12. These are also provided by the invention.
It has been found that Enterobacteriaceae produce L-threonine in an improved manner after over-expression of the mqo gene, which codes for malate:quinone oxidoreductase (E.C. 1.1.99.16).
According to the invention, it is also possible to use a DNA section which contains the DNA sequence of the gene of the malate:quinone oxidoreductase given in the databank of the National Center for Biotechnology Information (NCBI, Bethesda, Md., USA) with accession number P33940.
Alleles of the mqo gene which result from the degeneracy of the genetic code or due to xe2x80x9csense mutationsxe2x80x9d of neutral function can furthermore be used. It is also known that the amino acid methionine or formylmethionine coded by the start codon ATG can be removed in various proteins by enzymes of the host.
To achieve an over-expression, the number of copies of the corresponding genes can be increased, or the promoter and regulation region or the ribosome binding site upstream of the structural gene can be mutated. Expression cassettes which are incorporated upstream of the structural gene act in the same way. By inducible promoters, it is additionally possible to increase the expression in the course of fermentative L-threonine production. The expression is likewise improved by measures to prolong the life of the m-RNA. Furthermore, the enzyme activity is also increased by preventing the degradation of the enzyme protein. The genes or gene constructions can either be present in plasmids with a varying number of copies, or can be integrated and amplified in the chromosome. Alternatively, an over-expression of the genes in question can furthermore be achieved by changing the composition of the media and the culture procedure.
Instructions in this context can be found by the expert, inter alia, in Chang and Cohen (Journal of Bacteriology 134:1141-1156 (1978)), in Hartley and Gregori (Gene 13:347-353 (1981)), in Amann and Brosius (Gene 40:183-190 (1985)), in de Broer et al. (Proceedings of the National (sic) of Sciences of the United States of America 80:21-25 (1983)), in LaVallie et al. (BIO/TECHNOLOGY 11, 187-193 (1993)), in WO 98/04715, in Llosa et al. (Plasmid 26:222-224 (1991)), in Quandt and Klipp (Gene 80:161-169 (1989)), in Hamilton (Journal of Bacteriology 171:4617-4622 (1989), in Jensen and Hammer (Biotechnology and Bioengineering 58, 191-195 (1998) and in known textbooks of genetics and molecular biology.
Plasmid vectors which are capable of replication in Enterobacteriaceae, such as e.g. cloning vectors derived from pACYC184 (Bartolomxc3xa9 et al.; Gene 102, 75-78 (1991)), pTrc99A, which is described by Amann et al. (Gene 69:301-315 (1988)), or pSC101 derivatives (Vocke and Bastia, Proceedings of the National Academy of Science, USA 80 (21):6557-6561 (1983)) can be used. A strain transformed with a plasmid vector where the plasmid vector carries the nucleotide sequence which codes for the mqo gene can be employed in a process according to the invention.
In addition, it may be advantageous for the production of L-threonine with strains of the family Enterobacteriaceae to enhance, in particular to over-express, one or more enzymes of the known threonine biosynthesis pathway or enzymes of anaplerotic metabolism or enzymes for the production of reduced nicotinamide adenine dinucleotide phosphate, in addition to the mqo gene.
Thus, for example, one or more genes chosen from the group consisting of
the thrABC operon which codes for aspartate kinase, homoserine dehydrogenase, homoserine kinase and threonine synthase (U.S. Pat. No. 4,278,765),
the pyc gene which codes for pyruvate carboxylase (DE-A-19 831 609),
the pps gene which codes for phosphoenol pyruvate synthase (Molecular and General Genetics 231:332 (1992)),
the ppc gene which codes for phosphoenol pyruvate carboxylase (Gene 31:279-283 (1984)),
the genes pntA and pntB which code for transhydrogenase (European Journal of Biochemistry 158:647-653 (1986)),
the rhtB gene which imparts homoserine resistance (EP-A-0994190)
the rhtC gene which imparts threonine resistance (EP-A-1013765), and
the gdhA gene which codes for glutamate dehydrogenase (Gene 27:193-199 (1984))
can be enhanced, in particular over-expressed, at the same time.
It may furthermore be advantageous for the production of L-threonine, in addition to the enhancement of the mqo gene, for one or more of the genes chosen from the group consisting of:
the tdh gene which codes for threonine dehydrogenase (Ravnikar and Somerville, Journal of Bacteriology 169, 4716-4721 (1987)),
the mdh gene which codes for malate dehydrogenase (E.C. 1.1.1.37)
to be attenuated, in particular to be eliminated or for the expression thereof to be reduced at the same time.
Finally, in addition to enhancement of the mqo gene it may be advantageous for the production of L-threonine to eliminate undesirable side reactions, (Nakayama: xe2x80x9cBreeding of Amino Acid Producing Microorganismsxe2x80x9d, in: Overproduction of Microbial Products, Krumphanzl, Sikyta, Vanek (eds.), Academic Press, London, UK, 1982). Bacteria in which the metabolic pathways which reduce the formation of L-threonine are at least partly eliminated can be employed in a process according to the invention.
The microorganisms produced according to the invention can be cultured in the batch process (batch culture) or in the fed batch [sic] (feed process). A summary of known culture methods are [sic] described in the textbook by Chmiel (Bioprozesstechnik 1. Einfxc3xchrung in die Bioverfahrenstechnik [Bioprocess Technology 1. Introduction to Bioprocess Technology (Gustav Fischer Verlag, Stuttgart, 1991)) or in the textbook by Storhas (Bioreaktoren und periphere Einrichtungen [Bioreactors and Peripheral Equipment] (Vieweg Verlag, Braunschweig/Wiesbaden, 1994)).
The culture medium to be used must meet the requirements of the particular strains in a suitable manner. Descriptions of culture media for various microorganisms are contained in the handbook xe2x80x9cManual of Methods for General Bacteriologyxe2x80x9d of the American Society for Bacteriology (Washington D.C., USA, 1981).
Sugars and carbohydrates, such as e.g. glucose, sucrose, lactose, fructose, maltose, molasses, starch and optionally cellulose, oils and fats, such as e.g. soya oil, sunflower oil, groundnut oil and coconut fat, fatty acids, such as e.g. palmitic acid, stearic acid and linoleic acid, alcohols, such as e.g. glycerol and ethanol, and organic acids, such as e.g. acetic acid, can be used as the source of carbon. These substances can be used individually or as a mixture.
Organic nitrogen-containing compounds, such as peptones, yeast extract, meat extract, malt extract, corn steep liquor, soya bean flour and urea, or inorganic compounds, such as ammonium sulphate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate, can be used as the source of nitrogen. The sources of nitrogen can be used individually or as a mixture.
Phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts can be used as the source of phosphorus. The culture medium must furthermore comprise salts of metals, such as e.g. magnesium sulfate or iron sulfate, which are necessary for growth. Finally, essential growth substances, such as amino acids and vitamins, can be employed in addition to the abovementioned substances. Suitable precursors can moreover be added to the culture medium. The starting substances mentioned can be added to the culture in the form of a single batch, or can be fed in during the culture in a suitable manner.
Basic compounds, such as sodium hydroxide, potassium hydroxide, ammonia or aqueous ammonia, or acid compounds, such as phosphoric acid or sulfuric acid, can be employed in a suitable manner to control the pH. Antifoams, such as e.g. fatty acid polyglycol esters, can be employed to control the development of foam. Suitable substances having a selective action, e.g. antibiotics, can be added to the medium to maintain the stability of plasmids. To maintain aerobic conditions, oxygen or oxygen-containing gas mixtures, such as e.g. air, are introduced into the culture. The temperature of the culture is usually 25xc2x0 C. to 45xc2x0 C., and preferably 30xc2x0 C. to 40xc2x0 C. Culturing is continued until a maximum of L-threonine has formed. This target is usually reached within 10 hours to 160 hours.
The analysis of L-threonine can be carried out by anion exchange chromatography with subsequent ninhydrin derivatization, as described by Spackman et al. (Analytical Chemistry, 30, (1958), 1190), or it can take place by reversed phase HPLC as described by Lindroth et al. (Analytical Chemistry (1979) 51: 1167-1174).
A pure culture of the L-threonine-producing strain B-3996kurxcex94tdh/pVIC40, pMW218mqo was deposited on Jan. 24, 2001 at the Deutsche Sammlung fxc3xcr Mikrorgansimen [sic] und Zellkulturen (DSMZ=German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) as DSM 14004.
The process according to the invention is used for the fermentative preparation of amino acids, in particular L-threonine and L-isoleucine.
The present invention is explained in more detail in the following with the aid of embodiment examples.
The isolation of plasmid DNA from E. coli and all techniques of restriction, Klenow and alkaline phosphatase treatment are carried out by the method of Sambrook et al. (Molecular cloningxe2x80x94A laboratory manual (1989) Cold Spring Harbour Laboratory Press). Unless described otherwise, the transformation of E. coli is carried out by the method of Chung et al. (Proceedings of the National Academy of Sciences of the United States of America USA (1989) 86:2172-2175).
The incubation temperature during preparation of strains and transformants is 37xc2x0 C. Temperatures of 30xc2x0 C. and 44xc2x0 C. are used in the gene replacement process according to Hamilton et. al.